Molecular mechanism and history of non-sense to sense evolution of antifreeze glycoprotein gene in northern gadids

A fundamental question in evolutionary biology is how genetic novelty arises. De novo gene birth is a recently recognized mechanism, but the evolutionary process and function of putative de novo genes remain largely obscure. With a clear life-saving function, the diverse antifreeze proteins of polar fishes are exemplary adaptive innovations and models for investigating new gene evolution. Here, we report clear evidence and a detailed molecular mechanism for the de novo formation of the northern gadid (codfish) antifreeze glycoprotein (AFGP) gene from a minimal noncoding sequence. We constructed genomic DNA libraries for AFGP-bearing and AFGP-lacking species across the gadid phylogeny and performed fine-scale comparative analyses of the AFGP genomic loci and homologs. We identified the noncoding founder region and a nine-nucleotide (9-nt) element therein that supplied the codons for one Thr-Ala-Ala unit from which the extant repetitive AFGP-coding sequence (cds) arose through tandem duplications. The latent signal peptide (SP)-coding exons were fortuitous noncoding DNA sequence immediately upstream of the 9-nt element, which, when spliced, supplied a typical secretory signal. Through a 1-nt frameshift mutation, these two parts formed a single read-through open reading frame (ORF). It became functionalized when a putative translocation event conferred the essential cis promoter for transcriptional initiation. We experimentally proved that all genic components of the extant gadid AFGP originated from entirely non-genic DNA. The gadid AFGP evolutionary process also represents a rare example of the proto-ORF model of de novo gene birth where a fully formed ORF existed before the regulatory element to activate transcription was acquired.